Fluorescent molecules are widely used in chemistry and biochemistry. Fluorescent molecules are used to tag molecules for detection in a number of assays based on the creation or destruction of a fluorescent compound, providing a sensitive means to follow a reaction. For the most sensitive detection in a fluorescence-based system, the background signal should be small (i.e., little or no fluorescence) so there is an easily detectable signal. However, in common assays, the background fluorescence reduces the detection limit and sensitivity of the assay. An improved fluorescence assay is needed.
Prodrugs have been described which contain blocking groups which release the active drug upon activation with a suitable chemical trigger. For example, U.S. Pat. No. 6,030,997 describes a prodrug containing a pharmacologically active compound covalently bonded to a blocking group, whereby at pH 4 to 7, the covalent bond between the blocking group and pharmacologically active compound is broken, releasing the pharmacologically active compound. U.S. Pat. No. 5,672,584 describes the use in vivo of esterases which release peptides from cyclic peptide compounds. U.S. Pat. No. 5,965,119; continuation-in-part U.S. Pat. Nos. 6,303,569 and 6,214,330 describe double prodrug compounds having two cleavable groups—the first cleavable group is hydrolyzed and the second cleavable group undergoes the trialkyl lock lactonization reaction to release the desired compound. U.S. Pat. Nos. 6,413,507, 6,461,602 and 6,514,491 describe poly(ethylene glycol) compounds where a biologically active agent is linked to a poly(ethylene glycol) polymer through a hydrolyzable carbamate bond. The biologically active agent is released through hydrolysis of the carbamate bond.
PCT publication WO 03/099789 describes detection of enzymes using rhodamine derivatives substituted at one xanthylium amine group with a peptide which is protease cleavable and substituted at the other xanthylium amine group with a urea morpholine group. The bis-substituted derivative is reportedly not fluorescent. Upon exposure to the enzyme, the protease cleavable group is removed and the molecule is reported to become fluorescent. PCT publication WO 99/18856 describes detection of enzymes using rhodamine molecules substituted with specific enzyme-cleavable amino acid sequences. When contacted with an enzyme, the enzyme-cleavable amino acid sequences are removed from the rhodamine molecule and the rhodamine molecule reportedly shows an increase in fluorescence emission. PCT publication WO 03/062451 describes fluorescent compounds used to detect transport of target molecules across cell membranes. In WO 03/062451, the target molecule is linked to the fluorescent compound, making the compound non-fluorescent. The linkage between the compound and target molecule is cleaved upon entry into the cell membrane, resulting in compound fluorescence.
There is a need in the art for an improved fluorescence assay having blocking groups which release a fluorescent molecule upon activation with a trigger and optional urea-containing groups which can be used to attach desired groups to the fluorescent molecule.